In the course of a screen to identify mutants defective in the control of the sex-specific deaths of the CEM neurons using the reporter
pkd-2::gfp (see abstract by Schwartz and Horvitz), we found 29 independent isolates that had strong GFP expression in the pharynx, an organ that does not normally express this reporter. From this screen and further clonal and nonclonal screens, we have identified 68 mutants with the 'green pharynx' phenotype. This transgene misexpression is not dependent on chromosomal integration, high transgene copy number, or choice of co-injection marker, and the phenotype can be seen with at least one other GFP reporter that contains a different promoter. The green pharynx phenotype is dependent on vector sequence in the reporter construct, consistent with previous reports of a cryptic pharyngeal promoter in the Fire vectors. In the wild type, pharyngeal expression from this cryptic promoter can be inhibited by the inclusion of an insert in the reporter construct; in the context of some but not all inserts, this inhibition depends on a mechanism absent in the green pharynx mutants. We found that mutations in certain synthetic Multivulva (synMuv) genes (see abstracts by Andersen and Horvitz and by Harrison, Lu, and Horvitz) could produce the green pharynx phenotype. The synMuv genes act to inhibit vulval development. Animals mutant in two classes of synMuv gene (of the three classes, A, B, and C), but not animals mutant in one or more members of the same class, display a Multivulva phenotype. Several class B and class C synMuv genes have been cloned and shown to encode genes with homologs implicated in transcriptional modification and chromatin remodeling. The synMuv genes able to cause
pkd-2::gfp expression in the pharynx include one class A synMuv gene and three class B synMuv genes, a result that contrasts with the finding that genes in the A and B synMuv classes act separately and in parallel to prevent vulval cell fates. Mutations in 26 other synMuv genes have been tested and did not cause the green pharynx phenotype. 67 of the 68 mutations isolated based upon this phenotype appear to be alleles of three of these four synMuv genes; the fourth synMuv gene is maternally rescued for the green pharynx phenotype, and mutations in this gene were consequently not identified on the basis of this phenotype.
n3599 , the one green pharynx mutation that was not an allele of a synMuv gene, defined the gene
pag-6 ( pag , pattern of reporter gene expression abnormal).
pag-6(
n3599) mutants are not synMuv A, synMuv B, or synMuv C. Interestingly,
pag-6(
n3599) is synthetically lethal with a subset of class B synMuv mutations, including
lin-35 Rb . This subset does not correspond to subsets associated with other phenotypes, including the subset of class B synMuv mutants defective in regulation of the cell cycle (Boxem and van den Heuvel, Curr Biol 12: 906-11, 2002; Fay, Keenan, and Han, Genes and Development 16: 503-517, 2002). It is possible that the synMuv genes that are synthetically lethal with
pag-6(
n3599) share a normal function distinct from both vulval development and cell cycle regulation but nonetheless involving transcriptional repression.
pag-6(
n3599) causes altered function of a gene encoding a novel protein with homology to other worm proteins. Further investigation into these transgene-misexpression and synthetic-lethal phenotypes may define new transcriptional silencing complexes that include novel proteins and proteins previously implicated in silencing and that act in new combinations.